To identify viral determinants of dengue virus pathogenesis, this report describes the effects of naturally occurring dengue virus mutations on replication. The dengue virus type 2 structures probably involved in human virulence were defined previously by sequencing the complete genome of both American and SE Asian genotype templates in patient serum (37
). This genetic information was used to determine how American genotype virus structures influence replication in mosquito and human target cells. In the present study, we examined the effect of three American genotype structures on virus replication in vitro and demonstrated that American structures reduced virus output from human MDM and DC cultures. This change in replicative ability was specific for relevant human target cells since no alteration in replication was observed in mosquito cell cultures.
We hypothesized that changes in 5′- and 3′-end secondary structures and an amino acid charge change that are consistent in the American genotype viruses would decrease the replication efficiency of an SE Asian-derived infectious clone. Of the American structures examined, the E mutation (amino acid 390 Asn→Asp) was the only structure that by itself reduced virus output, albeit not significantly. Previous studies with MDM cultures suggested that the Asn at amino acid 390, in an infectious clone derived from strain New Guinea C (SE Asian genotype), is optimal for efficient replication (48
). Substitutions with a nonpolar (Ala), charged (Asp), or even a similar uncharged polar (Ser) side chain amino acid reduced virus output; however, the data were not normalized for any change in infectivity that might have occurred due to mutagenesis. Infectivity should be monitored since it has been shown that viral glycoproteins can affect tropism; for example, a single amino acid mutation at residue 160 of the Sindbis virus envelope glycoprotein E2 increases the efficiency of DC infection (13
). In the work shown here, the decreased dengue virus output measured was not due to alterations in the infectivity since virus output was determined as a function of the number of infected cells. It is possible that mutation of E 390 affects the efficiency of glycoprotein maturation and virion assembly, thereby reducing virus output. Although the E 390 Asn is not located in a glycosylation domain, other co- and posttranslational events may affect the efficiency of protein maturation (for a review, see reference 11
Results from the present study also indicated that changing E 390 to Asp was not sufficient to reduce virus output to the lower level measured for the wild-type American genotype dengue virus. Other changes were necessary to significantly reduce the dengue virus output from DC cultures. Sequences in ntr's have been shown to influence dengue virus replication. A single nucleotide substitution in the 5′ ntr of an SE Asian-derived virus, at nt 57, has been shown to decrease replication efficiency in mosquito cells and pathogenesis in mice (6
). Deletions in the 5′ ntr have been shown to affect serotype 4 dengue virus replication in cultured cells (7
). Construction of dengue viruses with alterations in the 3′ ntr resulted in viruses with restricted growth in vitro (44
). Efficient replication also requires interactions between the 5′ and 3′ termini (8
). Although nucleotide changes present in American genotype termini are not expected to disrupt long-range interactions, they were predicted to change RNA secondary structures at both ends compared to SE Asian ntr's (37
). Replacement of SE Asian with American terminal structures, either alone or in combination, did not reduce virus output but required the E mutation to significantly alter replication. One possible explanation for the requirement of the E mutation is that terminal structures decrease the replication or translation efficiency slightly, and this phenotype is more pronounced in the presence of the E mutation. In primary human foreskin fibroblasts, the replication of low-passage dengue type 2 viruses from Thailand generated less negative-strand RNA, suggesting a slightly reduced replication efficiency in comparison to 16681, whereas replication of Nicaraguan strains was blocked at a step prior to negative-strand synthesis (10
). It is possible that sequence differences within the ends of the Nicaraguan strains affect translation efficiency compared to other SE Asian genotype viruses. Results from our study demonstrate that substitution of SE Asian with multiple American genotype structures was necessary to reduce the replication efficiency to a level approaching that of a wild-type American genotype virus in human DCs. The mechanism by which viral replication is altered and the contribution of other conserved genotype structures on replication efficiency merits further study.
The correlation between SE Asian genotype structures and increased replicative ability in vitro underscores the correlation between viremia and pathogenesis. Prospective studies have shown that progression to DHF is associated with higher mean plasma viremias (39
). Progression to DHF has also been associated with increased levels of circulating NS1 (40
), also implying an increase in dengue virus replication. Three models can be proposed to explain the higher mean viremias observed for patients who develop DHF: (i) more cells are infected in DHF patients, thus generating more virus; (ii) more virus is produced per infected cell; or (iii) the virus is cleared at a slower rate compared to patients who develop DF, resulting in an increase in blood-borne virus. Little is known about the number of infected monocytes or DCs during human infection. In vitro experiments have shown that subneutralizing concentrations of antibody increased the number of dengue virus-infected monocytes in vitro, and this is referred to as ADE (4
). Data from dengue virus infection of primates indicated that increased viremias were associated with preexisting heterologous anti-dengue virus antibodies (17
), which may suggest that the increased viremia was due to an increase in the number of infected cells. ADE has been proposed as a mechanism for the increased risk of developing DHF associated with secondary dengue virus infection and may explain, in part, the increased viremias associated with DHF (5
). However, for dengue type 2 American genotype viruses, secondary infection is not associated with an increased risk of developing DHF (59
). Additionally, the likely initial targets for dengue virus replication are immature DCs in the skin (Langerhans cells) and infection efficiency is not influenced by the presence of anti-dengue virus antibodies, indicating that DCs are not susceptible to ADE (62
). We were unable to correlate the number of infected MDMs or DCs with genotype, although data from Table appear to suggest the opposite. In subsequent experiments, the number of infected cells varied considerably and sometimes the wild-type American genotype virus infected the highest percentage of cells. This may be due to infection rate variability observed between blood donors. Others have also reported that dengue virus infection rates vary among donors or cell lines and can be dependent on the differentiation state of cells (19
). A recent report compared the replication of four to eight SE Asian genotype isolates in different cell cultures and demonstrated that the number of infected cells varied between isolates in a human hepatoma cell line and primary human foreskin fibroblast cells (10
). Although we do not rule out the possibility of ADE playing a role in dengue virus pathogenesis, dengue virus infectivity appears to be quite variable and may hinder direct comparisons of the number of infected cells either in vivo or in vitro to the pathogenic potential of different dengue viruses.
With respect to the second proposed model that higher mean viremias observed for patients who develop DHF is the result of increased virus production per infected cell, our results may offer part of the explanation why SE Asian genotype viruses are associated with DHF and the indigenous American genotype viruses are not associated with DHF in the Western hemisphere. Since American genotype structures decreased virus output per infected cell, humans infected with the American strains may have lower mean viremias than patients infected with SE Asian genotype strains and thus do not develop DHF. The third proposed model that higher mean viremias are associated with slower virus clearance has already been addressed, and clinical data indicate that DHF was associated with faster virus clearance (58
). Although this may seem counterintuitive, this result is likely due to the association of DHF with secondary infections in Thailand and further supports the idea that the immune system is a factor in dengue virus pathogenesis.
In addition to viremia, elevated levels of other host factors have been shown to correlate with the development of DHF, including increased levels of liver enzymes, soluble adhesion molecules, and cytokines in plasma (28
). A number of reports have examined the production of immunomodulatory molecules after in vitro dengue virus infection of monocytes and endothelial cells (1
). Induction of these molecules initiates a number of cascades that potentially influence both proinflammatory and anti-inflammatory immune responses. In addition to monocytes, dengue virus infection of monocyte-derived DCs has been shown to induce maturation and activation, as well as the release of a number of cytokines (25
). Another virus that causes hemorrhagic fever, Hantaan, has also been shown to infect and activate DCs in vitro, resulting in the release of tumor necrosis factor alpha and alpha interferon (49
). Whether or not similar mechanisms are involved in the pathogenesis of DHF and Hantaan virus hemorrhagic fever with renal syndrome, the ability to infect target cells and modulate the immune system seems to be critical in the pathogenesis of both viruses. Our results suggest that, in comparison to American genotype viruses, some SE Asian genotype viruses replicate more efficiently. In turn, this could lead to the production of more virus and to increased infection of secondary sites resulting in increased levels of cytokines in plasma and more-severe disease symptoms. Clinical and immunologic data are needed from the Americas to determine whether there are distinguishing characteristics between the two genotypes that could be correlated with pathogenesis.
Replication in target cells is essential for dengue pathogenesis in the human host and is important for efficient transmission by the vector mosquito, Aedes aegypti
. From an ecologic or epidemiologic perspective, the SE Asian genotype virus appears to be displacing American genotype virus in this hemisphere (52
). This displacement could be the result of more efficient mosquito transmission by the SE Asian genotype viruses. Factors that influence mosquito transmission include the infection rate, the replicative ability, and the ability to disseminate to the salivary glands. Both host and viral determinants of dengue replication may affect viremia and thus contribute to the transmission potential of the virus to the mosquito. Some attenuated vaccine candidates have been shown to exhibit limited dissemination potential in mosquitoes (27
). Specifically, a 30-nt deletion in the 3′ ntr was shown to disrupt the ability of dengue virus to disseminate to the salivary gland after ingestion of a virus-spiked blood meal (57
). It has also been shown that a lower percentage of mosquitoes had a disseminated infection after ingestion of a blood meal spiked with American genotype viruses compared with those belonging to the SE Asian genotype (2
; P. M. Armstrong and R. Rico-Hesse, unpublished data). The ability to determine which American genotype structures influence infection and dissemination in the vector mosquito, thus providing an explanation for the displacement of the American genotype in this hemisphere, may be obtained by further utilizing the chimeric dengue viruses described here.
The mechanisms involved in severe dengue disease are complex and likely include viral and host determinants; these mechanisms may ultimately affect disease transmission and epidemiology. We have focused on identifying viral determinants associated with efficient replication in an attempt to correlate genotypic differences with the pathogenic potential of different viruses in this hemisphere. Our results indicate that American genotype structures decreased the replicative ability of an SE Asian-derived infectious clone and may provide part of the explanation of why the American genotype dengue viruses are not associated with the occurrence of DHF.